Enterobacter Cloacae Isolates Research Articles

Quantitative Analysis of Swertiamarin Content from Fagraea fragrans Leaf Extract using HPLC Technique and its Correlation to Antibacterial Activity

The rise in drug resistance poses escalating challenges for antibacterial medications, leading to an urgent demand for the exploration and innovation of new antibacterial drugs. Fagraea fragrans Roxb., belonging to the Gentianaceae family, is one of the common herbal medicines which can be found abundantly in Southeast Asia. The secoiridoid glucoside swertiamarin, one of the major compounds in F. fragrans leaf, exhibits antimicrobial effects. To guarantee the medicinal effectiveness of F. fragrans leaves, it is essential to identify a standardized analytical method for quantifying the active compound. In this study, the optimized HPLC method following ICH guideline was validated for the quantitative analysis of swertiamarin content in F. fragrans leaf in terms of linearity (y = 5733.5x - 369.1; R2 = 0.9999), accuracy (93.57-96.39% recovery), precision (0.91% RSD for repeatability precision; 1.19% RSD for intermediate precision), limit of detection (0.73 µg/mL), limit of quantitation (2.23 µg/mL), specificity (peak purity index = 0.999995) and robustness (% RSD <1).The maximum wavelength of swertiamarin was found to be at 238 nm. The amount of swertiamarin content in F. fragrans leaf extract conducted from the validated HPLC method was found to be 0.0259 ± 0.0005 g/100 g crude drug. The leaf extract exhibited antimicrobial activity against clinical isolates of Enterobacter cloacae, Klebsiella pneumonia, and Escherichia coli at 0.125 mg/mL, and Pseudomonas aeruginosa at 0.5, showing minimum inhibitory concentration (MIC) values. Whereas swertiamarin exhibited even lower MIC values. The developed HPLC analysis effectively determines swertiamarin content as a chemical marker to ensure the antimicrobial potential of F. fragrans leaves.

Scolopax rusticola Carrying Enterobacterales Harboring Antibiotic Resistance Genes.

The Eurasian woodcock (Scolopax rusticola) belongs to those bird species that make systematic migratory flights in spring and autumn in search of favorable breeding and wintering areas. These specimens arrive in the Mediterranean Area from northeastern European countries during the autumn season. The purpose of this study was to assess whether woodcocks can carry antibiotic resistance genes (ARGs) along their migratory routes. Although the role of migratory birds in the spread of some zoonotic diseases (of viral and bacterial etiology) has been elucidated, the role of these animals in the spread of antibiotic resistance has not yet been clarified. In this study, we analyzed the presence of beta-lactam antibiotic resistance genes. The study was conducted on 69 strains from 60 cloacal swabs belonging to an equal number of animals shot during the 2022-2023 hunting season in Sicily, Italy. An antibiogram was performed on all strains using the microdilution method (MIC) and beta-lactam resistance genes were investigated. The strains tested showed no phenotypic resistance to any of the 13 antibiotics tested; however, four isolates of Enterobacter cloacae and three of Klebsiella oxytoca were found to carry the blaIMP-70, blaVIM-35, blaNDM-5 and blaOXA-1 genes. Our results confirm the importance of monitoring antimicrobial resistance among migratory animals capable of long-distance bacteria spread.

The first report of the mobile colistin resistance gene, mcr-10.1, in Kenya and a novel mutation in the phoQ gene (S244T) in a colistin-resistant Enterobacter cloacae clinical isolate.

This study describes the identification of the mcr-10.1 gene in a clinical isolate of an ST1 Enterobacter cloacae isolate cultured in 2015 in Kenya. The isolate was multidrug resistant, phenotypically non-susceptible to various antibiotics, including colistin. Whole genome sequence analyses indicated carriage of chromosomally encoded antimicrobial resistance genes and the colistin-resistant gene mcr-10.1 located on a 72-kb plasmid designated pECC011b with an IncFIA(HI1) replicon directly adjacent to tyrosine recombinase gene, xerC, and downstream of an ISKPn26 insertion sequence. Studies have shown that expression of mcr-10.1 may not be sufficient to confer colistin resistance, but a novel non-synonymous mutation (S244T) was identified in the phoQ gene known to influence colistin resistance within lipid modification pathways, which could have complemented the mcr-10.1 resistance mechanism. In silico analysis of the mutant phoQ protein shows the location of the mutation to be at the Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases (HAMP) region, which plays a crucial role in the protein's activity. This study and our previous report of mcr-8 in Klebsiella pneumoniae indicate the presence of mobile mcr genes in the Enterobacterales order of bacteria in Kenya. The study points to the importance of regulation of colistin in the animal industry and enhancing surveillance in both human and animal health to curb the spread of mcr genes and accurately assess the risks posed by these mobile genetic elements in both sectors.IMPORTANCEThis paper reports the detection of new colistin resistance mechanisms in Kenya in a clinical isolate of Enterobacter cloacae in a patient with a healthcare-associated infection. The plasmid-mediated resistance gene, mcr-10.1, and a novel amino acid mutation S244T in the phoQ gene, located in a region of the protein involved in membrane cationic stability contributing to colistin resistance, were detected. Colistin is a critical last-line drug for multidrug-resistant (MDR) gram-negative human infections and is used for treatment and growth promotion in the animal industry. The emergence of the resistance mechanisms points to the potential overuse of colistin in the animal sector in Kenya, which enhances resistance, threatens the utility of colistin, and limits treatment options for MDR infections. This study highlights the need to enhance surveillance of colistin resistance across sectors and strengthen One Health policies that ensure antimicrobial stewardship and implementation of strategies to mitigate the spread of antibiotic resistance.

EVALUATION OF QUORUM SENSING SIGNALS OF STRONG BIOFILM PRODUCING BACTERIA VIA LC-MSMS, HPLC AND BIOSENSORS

This study aimed to show presence of Quorum Sensing (QS) signals of Gram-negative and Gram-positive biofilm producing bacteria isolated from real dairy process lines. Defining the profile and chemical composition of QS-signals is an important factor in control of microbial resistance and biofilm production. We especially focused on unusual behaviour of Gram-positive and Gram-negative isolates. Long-chain acyl-homoserine lactones (AHLs) signals (C14-HSL, C16-HSL and C18-HSL) and DFD (4,5-dihidroksi-2,3-pentanedione)-AI-2 signals of the isolates were studied by High-performance liquid chromatography (HPLC) and Liquid Chromatography with tandem mass spectrometry (LC-MSMS) methods. All Gram-positive isolates were defined as AHL-producers. All Gram-negative isolates, formerly defined as non-AHL producers by both biosensors and HPLC methods, were identified as AHL-producers. DFD signal was only detected from Gram-negative Klebsiella pneumonia, Enterobacter cloacae and Klebsiella oxytoca isolates. The results demonstrated that the QS-system is a complex system and biosensor microorganism may not be the best method for QS-signal identification. The results also provided new insights in defining the profile and chemical composition of QS-signals importance for interrupting the chemical communication completely to reduce biofilm formation and prevent resistance gain of microorganisms.

Isolation and Diagnosis of Bacteria in Bacteremia Patients and Study Their Resistance to Antibiotics in Kirkuk Hospitals

313 blood samples were collected from bacteremia patients, including 146 samples (30 from patients and 116 from outpatients) from Azadi teaching hospital, 36 samples from the dialysis unit at Kirkuk General Hospital, 126 samples (42 from inpatients and 84 from outpatients) from the Children's Hospital, and 5 samples from the Women's and Obstetrics Hospital in Kirkuk province, for the period from January 24, 2022, to September 10, 2022. The study, including the isolation and diagnosis of bacteria and the study of their resistance to antibiotics, The results show that 32 (17.87%) positive growth cultures were obtained from febrile patients, 3 (8.33%) from dialysis patients in the dialysis unit, and 15 (65.21%) from burn and wound patients. Fifty bacterial isolates were obtained, all of which were gram-positive. Staphylococcus was the highest with 28 isolates, including [(11) S.homoinis, (4) S.epidermidis epidermidis, (2) isolates each of S.haemolyticus and S. Wagner, and (9) Staphylococcus spp.], while Enterococcus faecalis was one isolate. The gram-negative bacteria were [(11) Pseudomonas aeruginosa, (5) Escherichia coli, (2) isolates of Enterobacter cloacae, and followed by one isolate of Raoultella terrigena, Acinetobacter spp., and Klebsiella spp.). Staphylococcus spp. resistance to 20 antibiotics was studied, and the species S.homoinis showed 100% resistance to (Oxacillin, Benzylpenicillin, and Amoxicillin). Whereas S.epidermidis epidermidis was 100% antibiotic-resistant (Oxacillin, Benzylpenicillin, and Amoxicillin). S. hemolyticus was resistant to (erythromycin, benzylbeniclin, amosiclin, amikachin, gentamicin, torramichin, and tetracycline) by 100%. S. warneri was resistant to (oxacillin, benzylpenicillin, amoxicillin, and dusidic Acid) at a rate of one hundred percent.

Mechanisms of ceftazidime/avibactam resistance in drug-naïve bacteraemic Enterobacterales strains without metallo-beta-lactamase production: Associated with ceftazidime impedance

In order to investigate ceftazidime/avibactam (CZA) resistance characteristics and mechanisms of bacteraemic Enterobacterales strains that had not been treated previously with CZA, 9708 strains were collected from 43 hospitals in 18 provinces across China from January 2019 to June 2020. The minimum inhibitory concentration (MIC) values of CZA in 165 (1.70%) strains were ≥8/4 mg/L. Ten (6.06%) CZA-resistant strains without metallo-β-lactamase production were obtained from the individuals without prior exposure to CZA, including six Escherichia coli isolates, three Klebsiella pneumoniae isolates and one Enterobacter cloacae isolate. Whole-genome sequencing revealed that ECB88611, ECB142593 and ECB144539 had encoded disrupted OmpF loss of function. OmpF of ECB126041 had a 2_9 MKRNILAV deletion; OmpK35 of three K. pneumoniae isolates harboured amino acid fragment deletions from positions 1 to 38; and ELB117287 had encoded disrupted OmpF. The G132D amino acid substitution of OmpC of ECB88611, ECB142593 and ECB144539, and the 134_135GD insertion of OmpK36 of three K. pneumoniae isolates were predicted to alter ceftazidime permeability. 333_334 YRIK or YRIN insertions occurred in PBP3 of six E. coli isolates. The relative expression of blaKPC-2 in KPB125108 was 4.527 ± 0.2166 times higher than the control strain, and the relative expression of acrF in six E. coli isolates was 2-3 times higher than the control strain. The addition of phenylalanine-arginine-β-naphthylamine at 100 mg/L decreased the MIC values of CZA against nine strains significantly. In conclusion, the antimicrobial resistance mechanisms in 10 isolates included increased expression of blaKPC-2, non-functional OMPs, upregulation of efflux pump activity, and variants of PBP3. Most of these mechanisms affected the antimicrobial activity of CZA by impeding ceftazidime.

Characterization of functional amyloid curli in biofilm formation of an environmental isolate Enterobacter cloacae SBP-8.

The biofilm formation by bacteria is a complex process that is strongly mediated by various genetic and environmental factors. Biofilms contribute to disease infestation, especially in chronic infections. It is, therefore important to understand the factors affecting biofilm formation. This study reports the role of a functional amyloid curli in biofilm formation at various abiotic surfaces, including medical devices, by an environmental isolate of Enterobacter cloacae (SBP-8) which has been known for its pathogenic potential. A knockout mutant of csgA, the gene encoding the major structural unit of curli, was created to study the effect of curli on biofilm formation by E. cloacae SBP-8. Our findings confirm the production of curli at 25°C and 37°C in the wild-type strain. We further investigated the role of curli in the attachment of E. cloacae SBP-8 to glass, enteral feeding tube, and foley latex catheter. Contrary to the previous studies reporting the curli production below 30°C in the majority of biofilm-forming bacterial species, we observed its production in E. cloacae SBP-8 at 37°C. The formation of more intense biofilm in wild-type strain on various surfaces compared to curli-deficient strain (ΔcsgA) at both 25°C and 37°C suggested a prominent role of curli in biofilm formation. Further, electron and confocal microscopy studies demonstrated the formation of diffused monolayers of microbial cells on the abiotic surfaces by ΔcsgA strain as compared to the thick biofilm by respective wild-type strain, indicating the involvement of curli in biofilm formation by E. cloacae SBP-8. Overall, our findings provide insight into biofilm formation mediated by curli in E. cloacae SBP-8. Further, we show that it can be expressed at a physiological temperature on all surfaces, thereby indicating the potential role of curli in pathogenesis.

Characterization of cephalosporin and fluoroquinolone resistant Enterobacterales from Irish farm waste by whole genome sequencing.

The Enterobacterales are a group of Gram-negative bacteria frequently exhibiting extended antimicrobial resistance (AMR) and involved in the transmission of resistance genes to other bacterial species present in the same environment. Due to their impact on human health and the paucity of new antibiotics, the World Health Organization (WHO) categorized carbapenem resistant and ESBL-producing as critical. Enterobacterales are ubiquitous and the role of the environment in the transmission of AMR organisms or antimicrobial resistance genes (ARGs) must be examined in tackling AMR in both humans and animals under the one health approach. Animal manure is recognized as an important source of AMR bacteria entering the environment, in which resistant genes can accumulate. To gain a better understanding of the dissemination of third generation cephalosporin and fluoroquinolone resistance genes between isolates in the environment, we applied whole genome sequencing (WGS) to Enterobacterales (79 E. coli, 1 Enterobacter cloacae, 1 Klebsiella pneumoniae, and 1 Citrobacter gillenii) isolated from farm effluents in Ireland before (n = 72) and after (n = 10) treatment by integrated constructed wetlands (ICWs). DNA was extracted using the MagNA Pure 96 system (Roche Diagnostics, Rotkreuz, Switzerland) followed by WGS on a MiSeq platform (Illumina, Eindhoven, Netherlands) using v3 chemistry as 300-cycle paired-end runs. AMR genes and point mutations were identified and compared to the phenotypic results for better understanding of the mechanisms of resistance and resistance transmission. A wide variety of cephalosporin and fluoroquinolone resistance genes (mobile genetic elements (MGEs) and chromosomal mutations) were identified among isolates that mostly explained the phenotypic AMR patterns. A total of 31 plasmid replicon types were identified among the 82 isolates, with a subset of them (n = 24), identified in E. coli isolates. Five plasmid replicons were confined to the Enterobacter cloacae isolate and two were confined to the Klebsiella pneumoniae isolate. Virulence genes associated with functions including stress, survival, regulation, iron uptake secretion systems, invasion, adherence and toxin production were identified. Our study showed that antimicrobial resistant organisms (AROs) can persist even following wastewater treatment and could transmit AMR of clinical relevance to the environment and ultimately pose a risk to human or animal health.

In vitro efficacy of cephamycins against multiple extended-spectrum β-lactamase-producing Klebsiella pneumoniae, Proteus mirabilis, and Enterobacter cloacae isolates from dogs and cats.

The susceptibility of 218 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates from companion animals to three cephamycins (cefmetazole, flomoxef, and latamoxef) was investigated. Phenotypic testing found 8 of 120 Klebsiella pneumoniae (KP) and 15 of 69 Enterobacter cloacae (EC) isolates were ESBL and AmpC β-lactamase (ABL) co-producers. Isolates of KP, Proteus mirabilis, and EC that only produced ESBL exhibited susceptibility rates to cefmetazole (95.5%, 82.7%, and 9.3%), flomoxef (99.1%, 96.6%, and 74.0%), and latamoxef (99.1%, 100%, and 100%), respectively. Notably, isolates of KP and EC co-producing ESBL and ABL had significantly lower susceptibility rates to the studied drugs when compared with only ESBL producers. This implies that the in vitro activity of cephamycins against ESBL-producing bacteria can differ depending on ABL production and bacterial species.

Variation in antibiotic resistance patterns for children and adults treated at 166 non-affiliated US facilities using EHR data.

Antibiotic resistance (AR) is a global public health threat. Surveillance of baseline AR and trends and emerging resistance among priority bacterial isolates with respect to the age of the patients and the type of healthcare setting are required due to differences in antimicrobial need and use in these populations. We performed a retrospective study using deidentified electronic health record (EHR) data in the Cerner Health Facts™ data warehouse. Antibiotic susceptibility data were extracted for all bacterial isolates of interest at 166 non-affiliated healthcare facilities reporting microbiology susceptibility results of the FDA recommended antibiotics between the years 2012 to 2017. We assessed and visualized the slope coefficient from linear regression to compare changes in resistance over time for the four patient care groups. The trends in resistance rates to clinically relevant antibiotics were influenced by age and care setting. For example, ertapenem-resistant Enterobacter cloacae isolates from children overall increased significantly compared with adults (0.7% to 9.8%, 2.1% to 2.8%, P = 0.00013) and isolates from children in paediatric facilities increased significantly compared with facilities treating adults and children (0.1% to 27.1%, 0.9% to 3.8%, P = 0.0002). Large-scale analysis of EHR data from 166 facilities shows that AR patterns for some bug-drug combinations vary by care setting and patient age. We describe novel data visualizations to interpret large-scale EHR data on the prevalence and trends of AR that should influence antimicrobial prescribing and antimicrobial stewardship programme interventions.

THE PREVALENCE OF BLANDM, BLAVIM GENES AMONG ENTEROBACTER CLOACAE BACTERIA

This study was aimed to detect the prevalence of blaVIM and blaNDM genes among Enterobacter cloacae isolates that were isolated from Iraqi patients. About 50 bacterial isolates were collected from different hospitals in Baghdad city and all these isolates were diagnosed using biochemical tests and CHROMagar culture media and conformed using the Vitek II system. The antibiotic susceptibility for E.cloacae isolates was determined using the disk diffusion (Kirby Bauer) method and the results showed that these bacteria were showed resistance to Cefepime and Meropenem antibiotics in percentage (50%), (40%) respectively. In addition it is resistant to Amikacin, ampicillin/sulbactam, Piperacillin, and aztreonam was (20%), (50%), (40%), and (55%) respectively. Also it has been noted that all isolates showed resistance to Cefixime in percentage (100%). EDTA combined disc test was used to detect the ability of E. cloacae isolates to produce carbapenemase and the results were showed that only 8 isolates were gave positive results , While the prevalence of blaVIM and blaNDM was determined using the conventional Polymerase chain reaction technique. And the result showed that only four isolates harbored blaNDM while blaVIM was present in 3 isolates

Efficacy of diversely isolated lytic phages against multi-drug resistant Enterobacter cloacae isolates in Kenya.

BackgroundEnterobacter cloacae causes nosocomial infections in 15% of patients in low- and middle-income countries with emergence of carbapenem resistance. The utilisation of bacteriophages for therapeutic purposes is crucial for eradicating these resistant bacterial strains.ObjectiveThis study evaluated the efficacy of lytic phages on bacterial isolates of E. cloacae and determined their stability in various physicochemical conditions.MethodsTwenty-nine lytic phages were isolated from the waste water of six informal settlements in Nairobi County, Kenya, from July 2019 to December 2020 and cross-reacted with 30 anonymised clinical isolates of E. cloacae. Six phages were then selected for physicochemical property studies. Phages were described as potent upon lysing any bacterial strain in the panel.ResultsSelected phages were stable at 4 °C – 50 °C with a 5.1% decrease in titre in four of six phages and a 1.8% increase in titre in two of six phages at 50 °C. The phages were efficient following two weeks incubation at 4 °C with optimal activity at human body temperature (37 °C) and an optimal pH of 7.5. Phages were active at 0.002 M and 0.015 M concentrations of Ca2+ ions. The efficiency of all phages decreased with increased exposure to ultraviolet light. All phages (n = 29) showed cross-reactivity against anonymised clinical isolates of E. cloacae strains (n = 30). The most potent phage lysed 67.0% of bacterial strains; the least potent phage lysed 27.0%.ConclusionThis study reveals the existence of therapeutic phages in Kenya that are potent enough for treatment of multi-drug resistant E. cloacae.

Insights Into the Dynamics and Composition of Biofilm Formed by Environmental Isolate of Enterobacter cloacae.

Bacterial biofilms are clinically admissible and illustrate an influential role in infections, particularly those related to the implant of medical devices. The characterization of biofilms is important to understand the etiology of the diseases. Enterobacter cloacae are known for causing infections by forming biofilms on various abiotic surfaces, such as medical devices. However, a detailed characterization in terms of morphology and the molecular composition of the formed biofilms by this bacterium is sparse. The present study provides insights into the biofilm formation of E. cloacae SBP-8, an environmental isolate, on various surfaces. We performed assays to understand the biofilm-forming capability of the SBP-8 strain and characterized the adhering potential of the bacteria on the surface of different medical devices (foley latex catheter, enteral feeding tube, and glass) at different temperatures. We found that medical devices exhibited strong colonization by E. cloacae SBP-8. Using field emission-scanning electron microscopy (FE-SEM) studies, we characterized the biofilms as a function of time. It indicated stronger biofilm formation in terms of cellular density and EPS production on the surfaces. Further, we characterized the biofilm employing surface-enhanced Raman spectroscopy (SERS) and identified the vast heterogenic nature of the biofilm-forming molecules. Interestingly, we also found that this heterogeneity varies from the initial stages of biofilm formation until the maturation and dispersion. Our studies provide insights into biofilm composition over a period of time, which might aid in understanding the biofilm dispersion phases, to enhance the presently available treatment strategies.

Multicenter surveillance of antimicrobial susceptibilities and resistance mechanisms among Enterobacterales species and non-fermenting Gram-negative bacteria from different infection sources in Taiwan from 2016 to 2018

To explore the invitro antimicrobial susceptibility among clinically important Gram-negative bacteria (GNB) in Taiwan. From 2016 through 2018, a total of 5458 GNB isolates, including Escherichia coli (n=1545), Klebsiella pneumoniae (n=1255), Enterobacter species (n=259), Pseudomonas aeruginosa (n=1127), Acinetobacter baumannii complex (n=368), and Stenotrophomonas maltophilia (n=179), were collected. The susceptibility results were summarized by the breakpoints of minimum inhibitory concentration (MIC) of CLSI 2020, EUCAST 2020 (for colistin), or published articles (for ceftolozane/tazobactam). The resistance genes among multidrug-resistant (MDR) or extensively drug-resistant (XDR)-GNB were investigated by multiplex PCR. Significantly higher rates of non-susceptibility (NS) to ertapenem and carbapenemase production, predominantly KPC and OXA-48-like beta-lactamase, were observed in Enterobacterales isolates causing respiratory tract infection than those causing complicated urinary tract or intra-abdominal infection (12.7%/3.44% vs. 5.7%/0.76% or 7.7%/0.97%, respectively). Isolates of Enterobacter species showed higher rates of phenotypic extended-spectrum β-lactamase and NS to ertapenem than E. coli or K. pneumoniae isolates. Although moderate activity (54-83%) was observed against most potential AmpC-producing Enterobacterales isolates, ceftolozane/tazobactam exhibited poor invitro (44.7-47.4%) activity against phenotypic AmpC Enterobacter cloacae isolates. Additionally, 251 (22.3%) P. aeruginosa isolates exhibited the carbapenem-NS phenotype, and their MDR and XDR rate was 63.3% and 33.5%, respectively. Fifteen (75%) of twenty Burkholderia cenocepacia complex isolates were inhibited by ceftolozane/tazobactam at MICs of ≤4μg/mL. With the increase in antibiotic resistance in Taiwan, it is imperative to periodically monitor the susceptibility profiles of clinically important GNB.

Molecular Epidemiology, Microbial Virulence, and Resistance of Carbapenem-Resistant Enterobacterales Isolates in a Teaching Hospital in Guangzhou, China.

Infection caused by carbapenem-resistant Enterobacterales (CRE) is a global public health problem. We performed whole-genome sequencing to investigate the molecular epidemiological characteristics of local CRE infections and understand the prevalence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (CRKP). Analysis of multiLocus sequence typing (MLST), antibiotic resistance genes, plasmid replicons, virulence genes, and the genetic environment was also performed. Klebsiella pneumoniae (89, 60.95%) was the most common CRE species, primarily prevalent in the intensive care unit (36, 40.45%). Most CRE strains showed a high resistance rate to multiple antibiotics, especially cephalosporins and carbapenems. However, most of these isolates were susceptible to tigecycline (81.7%). Notably, the predominant sequence type (ST) of CRKP isolates was ST11 (80.90%, 72/89), with 93.05% as Klebsiella pneumoniae carbapenemase (KPC)-ST11. In Escherichia coli isolates, ST410 (21.43%, 6/28) was the predominant type, with approximately half carrying blaNDM-5, and importantly, the ST167 carbapenem-resistant Escherichia coli (CRECO) harbors both New Delhi metallo-β-lactamase (NDM)-5 and KPC-2. In Enterobacter cloacae isolates, three cases of ST88 were carrying the blaNDM-1 gene, and the ST594 carbapenem-resistant Enterobacter cloacae (CRECC) carrying NDM-1 and KPC-2 has also been identified. In addition, we found three novel STs, ST5386-ST5388. The IncFII (pHN7A8) (98.41%, 62/63) was the most common plasmid replicon type in KPC-2-producing CRKP strains, and the predominant plasmid ST of IncF was [f33:A-:B-] (n = 73). Two CRKP isolates were found to carry 4 virulence genes (iutA, iroB, rmpA, and rmpA2). As concluded, among CRKP strains, ST11 was the predominant ST with blaKPC-2, and a large proportion of CRKP strains co-harbor blaKPC-2, blaSHV, blaCTX-M, blaTEB-1B, and fosA. The predominant carbapenemase genes carried by CRECO and CRECC were blaNDM-1 and blaCTX-M, respectively.

The Emergence of Carbapenem Resistant Enterobacteriaceae Producing GIM-1 and SIM-1 Clinical Isolates in Khartoum-Sudan.

PurposeThe aim of this study was to detect multidrug resistant GIM-1 and SIM-1 producing Enterobacteriaceae clinical isolates from hospitalized patients across three Khartoum State Teaching Hospitals, Sudan.Patients and MethodsFrom May 2018 to October 2019, Enterobacteriaceae clinical isolates from inpatients admitted to different Khartoum state hospitals. Genes for carbapenemase (GIM-1 and SIM-1) were amplified by polymerase chain reaction (PCR). Agar dilution method was used to determine MICs for imipenem and meropenem after antimicrobial susceptibility testing.ResultsFive (1.29%) isolates of Enterobacteriaceae [2 (0.51%) Escherichia coli isolates produce GIM-1, 2 (0.51%) Klebsiella pneumoniae isolates (one [0.25%] of each produce of GIM-1 and of SIM-1), and 1 (0.25%) Enterobacter cloacae isolate produce GIM-1]. Susceptibility profiling of the isolates showed a low-level resistance to imipenem and meropenem MICs (8, 16 and 32 μg/mL). It also had resistance to ampicillin, extended-spectrum cephalosporin’s, aztreonam, and amoxicillin-clavulanate and with the two K. pneumoniae strains showing resistance to colistin.ConclusionWe report the emergence of four GIM-1 producing Enterobacteriaceae strains and one strain of SIM-1 producing K. pneumoniae genes, isolated from hospitalized patients, with a high resistance pattern to antimicrobial agents. Whole-genome sequencing (WGS) is necessary for precise identification of clonal diversity backgrounds of acquired carbapenemase genes in diagnostic laboratories as the number of cases of carbapenem resistant Enterobacteriaceae infection increases annually.

Antimicrobial Activity Profiles and Potential Antimicrobial Regimens against Carbapenem-Resistant Enterobacterales Isolated from Multi-Centers in Western Thailand.

The spread of carbapenem-resistant Enterobacterales (CRE) constitutes a global health burden. Antimicrobial susceptibility and types of carbapenemase differ by geographic region. This study aimed to (1) examine the minimum inhibitory concentrations (MICs) and antibiotic resistance genes and (2) investigate antibiotic dosing regimens against CRE using Monte Carlo simulation. Clinical carbapenem-resistant Klebsiella pneumoniae (CRKP), Escherichia coli (CREC), and Enterobacter cloacae (CREclo) isolates were collected from various hospitals in western Thailand. Broth microdilution was performed, and the types of carbapenemase and mcr-1 genes were detected using polymerase chain reaction (PCR). Monte Carlo simulation was used to establish optimal antimicrobial dosing regimens meeting the criterion of a cumulative fraction of response (CFR) >90%. A total of 150 CRE isolates from 12 hospitals were included. The proportion of CRKP (76%) was greater than that of CREC (22%) and CREclo (2%). Regional hospitals reported higher rates of resistance than general hospitals. Most isolates were resistant to aztreonam and ceftazidime/avibactam, whereas they were highly susceptible to aminoglycosides. Most carbapenemases were NDM (47.33%), OXA-48 (43.33%) and NDM plus OXA-48 (6.67%); five OXA-48 positive isolates carried mcr-1 genes. Currently, high-dose tigecycline is the only optimal regimen against CRE isolates. Further extensive research on antibiotic synergism or new antibiotics should be conducted.

Identification of Plasmid-Mediated Tigecycline-Resistant Gene tet(X4) in Enterobacter cloacae from Pigs in China.

ABSTRACTTwo tet(X4)-positive Enterobacter cloacae isolates TECL_1 and TECL_2 were isolated from pigs in China. S1-PFGE and Southern blotting showed that tet(X4) located on plasmids in the size of ∼290 kb and ∼190 kb in TECL_1 and TECL_2, respectively. Conjugation experiment demonstrated that the tet(X4)-harboring plasmid can transfer from the donor strain TECL_1 and TECL_2 to the recipient strain Escherichia coli J53, and the tigecycline resistance of transconjugants was increased by 128-fold and 64-fold compared with E. coli J53, respectively. We obtained the complete plasmid sequence of pTECL_2-190k-tetX4 (190,185 bp) from E. cloacae TECL_2 and found that the plasmid was a hybrid plasmid with replicon types of IncFIA, IncHI1A and IncHI1B. We further analyzed 85 tet(X4)-carrying plasmids in the public database and clarified that pTECL_2-190k-tetX4-like plasmid was widespread in multiple species of Enterobacteriaceae.IMPORTANCE We identified two tet(X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B. By comparing all the known tet(X4)-carrying plasmids, we found that pTECL_2-190k-tetX4-like plasmid has been disseminated across various species in China. Our study expanded the identification of tet(X4)-positive species and emphasized that pTECL_2-190k-tetX4-like plasmid has spread widely in various species.

Blood culture surveillance in a secondary care hospital in Benin: epidemiology of bloodstream infection pathogens and antimicrobial resistance

BackgroundAlthough global surveillance of antimicrobial resistance (AMR) is considered key in the containment of AMR, data from low- and middle-income countries, especially from sub-Saharan Africa, are scarce. This study describes epidemiology of bloodstream infections and antimicrobial resistance rates in a secondary care hospital in Benin.MethodsBlood cultures were sampled, according to predefined indications, in BacT/ALERT FA Plus and PF Plus (bioMérieux, Marcy-l’Etoile, France) blood culture bottles (BCB) in a district hospital (Boko hospital) and to a lesser extent in the University hospital of Parakou. These BCB were incubated for 7 days in a standard incubator and twice daily inspected for visual signs of growth. Isolates retrieved from the BCB were processed locally and later shipped to Belgium for reference identification [matrix-assisted laser desorption/ionization time-of-flight spectrometry (MALDI-TOF)] and antibiotic susceptibility testing (disk diffusion and E-tests).ResultsFrom October 2017 to February 2020, 3353 BCB were sampled, corresponding to 3140 blood cultures (212 cultures consisting of > 1 BCB) and 3082 suspected bloodstream infection (BSI) episodes. Most of these cultures (n = 2471; 78.7%) were sampled in children < 15 years of age. Pathogens were recovered from 383 (12.4%) cultures, corresponding to 381 confirmed BSI. 340 of these pathogens were available and confirmed by reference identification. The most common pathogens were Klebsiella pneumoniae (n = 53; 15.6%), Salmonella Typhi (n = 52; 15.3%) and Staphylococcus aureus (n = 46; 13.5%). AMR rates were high among Enterobacterales, with resistance to third-generation cephalosporins in 77.6% of K. pneumoniae isolates (n = 58), 12.8% of Escherichia coli isolates (n = 49) and 70.5% of Enterobacter cloacae isolates (n = 44). Carbapenemase production was detected in 2 Escherichia coli and 2 Enterobacter cloacae isolates, all of which were of the New Delhi metallo-beta lactamase type. Methicillin resistance was present in 22.4% of S. aureus isolates (n = 49).ConclusionBlood cultures were successfully implemented in a district hospital in Benin, especially among the pediatric patient population. Unexpectedly high rates of AMR among Gram-negative bacteria against commonly used antibiotics were found, demonstrating the clinical and scientific importance of clinical bacteriology laboratories at this level of care.

Diversity of cellulolytic bacteria from Macrotermes gilvus gut isolated from Indralaya peatland region, Indonesia

Abstract. Oktiarni D, Kasmiarti G, Nofyan E, Miksusanti, Hasanudin, Hermansyah. 2021. Diversity of cellulolytic bacteria from Macrotermes gilvus gut isolated from Indralaya peatland region, Indonesia. Biodiversitas 23: 486-495. The termite Macrotermes gilvus Hagen from Tanjung Senai, Inderalaya peatland region, is classified as a high-level termite. Termites like eusocial insects break down cellulosic biomass into glucose by bacteria that produce cellulolytic enzymes in their gut. In the present study, new species of cellulolytic bacteria from Macrotermes gilvus Hagen gut were determined by biomolecular assay. Bacterial isolates were isolated and purified by using DNA kit and the optical density of DNA bacterial isolates was obtained by using spectrophotometer nanodrop. A total of 24 bacterial isolates were amplified using PCR to determine the fragment of 16S rRNA gene, performed using BLAST-N program and compared with NCBI database. Result from the sequences of 16S rRNA gene showed that the new species were identify as Enterobacter cloacae (isolates 1, 3, 6, 7, 10, 14, 19, 20, 25, 28, 39, 40, 44, 60, and 64), Klebsiella pneumoniae (isolates 24, 43, and 62), Klebsiella quasipneumoniae (isolates 29, 36, and 37), Klebsiella varicolla (isolate 52), Enterobacter roggenkampii (isolate 56) and Enterobacter asburiae (isolate 59).